Method and apparatus for determining nutrient stimulation of biological processes

ABSTRACT

A method and apparatus for determining the nutrients to stimulate microorganisms in a particular environment. A representative sample of microorganisms from a particular environment are contacted with multiple support means wherein each support means has intimately associated with the surface of the support means a different nutrient composition for said microorganisms in said sample. The multiple support means is allowed to remain in contact with the microorganisms in the sample for a time period sufficient to measure difference in microorganism effects for the multiple support means. Microorganism effects for the multiple support means are then measured and compared. The invention is particularly adaptable to being conducted in situ. The additional steps of regulating nutrients added to the particular environment of microorganisms can enhance the desired results. Biological systems particularly suitable for this invention are bioremediation, biologically enhanced oil recovery, biological leaching of metals, and agricultural bioprocesses.

CONTRACTUAL ORIGIN OF THE INVENTION

The United States Government has rights in this invention disclosedunder Contract Number DE-AC07-76ID01570 between the U.S. Department ofEnergy and EG&G Idaho, Inc., now Contract Number DE-AC07-94ID13223 withLockheed Idaho Technologies Company.

This application is a continuation of application Ser. No. 08/448,022filed May 23, 1995, now U.S. Pat. No. 5,686,299.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates generally to a method and apparatus fordetermining nutrient stimulation of biological processes. Suchbiological processes include bioremediation, biologically enhanced oilrecovery, biological leaching of metals, and agricultural bioprocessessuch as biological treatment of compost.

2. Background Art

Many biological processes depend upon nutrients to stimulate growth andactivity of the microorganisms involved. These nutrients can be organicand/or inorganic materials. Organic materials such as acetate orcitrates provide carbon for building new cell material or biomass,and/or energy. Inorganic materials such as nitrates and phosphatesprovide the nitrogen and phosphorous which are key building blocks forthe cells.

An example of the role nutrients play in bioremediation processes Is inthe soil vapor extraction processes for remediating ground watercontaminated with organic and inorganic materials. The processesdescribed in Billings et al, U.S. Pat. No. 5,221,159 are illustrative.Billings et al, describes methods and processes for in situ removal ofcontaminants, such as organic and inorganic products, from soil andgroundwater by providing one or more injection wells drilled to a depthbelow the water table and an extraction well drilled to a depth abovethe water table. Oxygenated gas is injected under pressure through theinjection well while vacuum is applied to the extraction well. Most ofthe contaminants removed from the groundwater and vadose zone are due tobiochemical processes. Microbes from the contaminated site are extractedand analyzed to determine the genera present in the samples. Microbesfrom genera known to be useful in biodegrading the contaminants are thenisolated, and the isolated microbes are fermented to increase thenumbers of useful organisms. Then the fermented microorganisms arereintroduced through the injection or extraction wells to enhancebiodegradation. If necessary, because of low levels of contaminants andconsequent low levels of microbes, nutrients are provided to themicrobial population to sustain high levels of degradation activity.This method suffers from not knowing, except through trial and error,what nutrient composition should be added for the particular environmentinvolved to enhance the in situ bioremediation that is key to thesuccess of this technology.

A paper presented by the Cullimore et al at the First InternationalSymposium on Microbiology of the Deep Subsurface in Jan. 15-19, 1990,entitled "Development Strategies for the Utilization of In-WellIncubation Devices (IWID) to Establish Management Strategies for theBioremediation of Chemically Impaired Water Wells and GroundwaterSystems" notes the problem of identifying the proper nutrientcomposition to facilitate the desired microbial activity inbioremediation processes. The solution proposed in this paper is to puta group of test tubes containing different nutrient compositions intothe borehole and then adding samples of the contaminated water into thedifferent test tubes. After a one month incubation period the test tubesare removed from the borehole and analyzed.

The Cullimore et al process for determining nutrient addition tostimulate microbial activity suffers from significant problems. First ofall the process uses test tubes which measure only the free living(unattached) microbial communities. However, the free living microbialpopulations are in many systems much less significant than the attachedto microbial populations. One study is believed to have shown thatbiological activity from attached microbial populations was 100 timesmore significant than that due to free living microbial communities.

Another significant draw-back of the Cullimore et al approach is thatthe test is essentially a batch culture and thus the test would onlyrepresent the chemical conditions of the particular environment at thebeginning of the test. Therefore, there are serious concerns about thecommercial applicability of the Cullimore et al test.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method andapparatus for determining nutrient stimulation of biological processes.

It is also an object of the invention to provide a method and apparatusfor determining nutrient stimulation for attached microbial populationsin a particular environment.

It is another object of the invention to provide a method and apparatusfor in situ determination of nutrients suitable for stimulation of adesired microbial population in a particular biological system.

It is still another object of the invention to enhance the desiredresult of a particular biological process by regulating the nutrientcomposition added to such biological process in response to results fromtests performed as to the effect of different nutrient compositionadditions to the biological process.

A device for determining nutrient limitations on growth and activity ofmicroorganisms in an aqueous environment comprising holding means forholding a liquid nutrient medium for stimulating growth of themicroorganisms, the holding means including nutrient-diffusing substratemeans disposed on the holding means for gradually releasing an effectiveamount of the liquid nutrient medium such that the microorganisms arestimulated to attach to and grow on the nutrient-diffusing substratemeans; and suspension means disposed on the holding means for suspendingthe device in the aqueous environment. The device preferably comprises aplurality of the holding means coupled together and spaced apart in alinear series by connecting means. Preferably the holding meanscomprises an impermeable cylindrical side wall, an impermeable topdisposed on the side wall, and a porous bottom disposed on the sidewall, the porous bottom comprising the nutrient-diffusing substratemeans and the side wall, top, and bottom defining a cavity for holdingthe liquid nutrient medium.

The device preferably comprises means, such as a removable plug, forfilling the cavity with the liquid nutrient medium. Also the porousbottom may be detachable from the side wall, and additionally the devicemay comprise means for sealing the porous bottom to the side wall.

The device preferably further comprises a gas sensor disposed thereonfor monitoring a gas, such as oxygen and/or carbon dioxide, given off bythe microorganisms attached to and growing on the nutrient-diffusingsubstrate means. The device may also comprise a computer linked to thegas sensor for receiving monitoring data generated by the gas sensor,which data may then be used to have the computer control the addition ofnutrient composition to the biological system from which the data wasgenerated.

A preferred device is one where the nutrient-diffusing substrate meanscomprises a porous ceramic support.

A method of determining nutrient limitations on growth of microorganismsin an aqueous environment comprising the steps of providing a devicecomprising holding means for holding a liquid nutrient medium forstimulating growth of the microorganisms, the holding means includingnutrient-diffusing substrate means disposed on the holding means forgradually releasing an effective amount of the liquid nutrient mediumsuch that the microorganisms are stimulated to attach to and grow on thenutrient-diffusing substrate means; and suspension means disposed on theholding means for suspending the device in the aqueous environment;placing a liquid nutrient medium to be tested in the holding means;suspending the device in the aqueous environment for a sufficient timefor the nutrient-diffusing substrate means to gradually release theliquid nutrient medium such that the microorganisms attach to and growon the nutrient-diffusing substrate means; and measuring growth of themicroorganisms on the nutrient-diffusing substrate means. The method isespecially appropriate when the aqueous environment comprises anaquifer.

As used herein, "aqueous environment" means an aqueous locale, such asan aquifer or water well, that a person skilled in this art might selectfor determining nutrient limitations on growth of microorganismstherein. Such microorganisms could be indigenous or introduced. Thegrowth of such microorganisms would be important in biological processessuch as bioremediation, biologically enhanced oil recovery, biologicalleaching of metals, biological treatment of compost, and the like.

As used herein, "liquid nutrient medium" means an aqueous solutioncontaining a carbon source and energy source for supporting growth ofmicroorganisms. These carbon and energy sources are not considered to benovel, but are conventional carbon sources and energy sources known inthe art. Such carbon and energy sources include, without limitation,carbohydrates, organic acids, alcohols, nitrogenous compounds,phosphorous compounds, and the like.

As used herein, "effective amount" means an amount of a liquid nutrientmedium sufficient to stimulate growth of microorganisms indigenous to orintroduced to an aqueous environment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of a component of an apparatusaccording to the present invention, comprising a support means and acontainer, to be filled with a nutrient solution, to be tested forpossible application to a biological process, according to a preferredembodiment of the invention.

FIG. 2 is a view of the apparatus of this present invention comprisingmultiple components such as the one shown in FIG. 1, attached to eachother, according to a preferred embodiment of the present invention.

FIG. 3 is a graph showing the diffusion of nitrate from a component suchas shown in FIG. 1 of the invention under batch conditions with nomicroorganisms present.

FIG. 4 is a graph showing the relationship of biomass produced on theporous surface of a component such as shown in FIG. 1 relative to thenutrients supplied within the component.

FIG. 5 is a graph showing the results of principal components analysiswhich compares the results obtained from attached microbial communitiesto free-living microbial communities from the same aquifer.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to FIG. 1, the apparatus in its preferred embodimentcomprises a cylinder 10, preferably cylindrical in shapes with anonporous lid 12 and a porous bottom surface 14, that, when assembledwith 10, form a container with a cavity 16. The assembled unit is madeto hold a solution or gel in cavity 16 which contains one or morenutrients that are intended to diffuse through the porous surface 14.The nonporous lid 12 and cylinder 10 may be machined separately or froma single unit. An "O" ring 18 seals the gap between the cylinder 10 andthe porous surface 14 and the porous surface is secured to the cylinderusing bolts 20. A plug 21 is present in the nonporous lid 12 to providea hole through which the solution or gel can be poured. The entireassembly is composed of materials that can be sterilized, preferably inan autoclave, prior to use. The dimensions of the assembled unit must besuch that it can fit into the borehole of interest, allowing it to besuspended beneath the level of the water table.

The porous surface 14 is the surface upon which microorganisms grow orare active and such microorganisms can be monitored using sensorsattached to porous base 14 or after the device is contacted with arepresentative sample for a period of time sufficient to measuredifferences in the microorganism effects. The porous surface 14 can bescraped to acquire and measure the microorganisms that have becomeattached.

In FIG. 2. several of the preferred components shown in FIG. 1. areconnected in sequence as they would be when suspended by a cable to aposition beneath the level of the water table in an aquifer. Incubationwithin the aquifer occurs in this manner for a period of time necessaryto measure differences in the microorganisms on the porous surface ofthe different components. Each of the preferred embodiments contains adifferent nutrient or different concentrations of the same nutrient.

The invention is further Illustrated by the following non-limitingexamples.

EXAMPLE 1

The concentration of nitrate measured with respect to time in replicated2 liter vessels similar to FIG. 1, each containing sterile aquifer waterand a single nitrate-containing nutrient composition is depicted in FIG.3. Nitrate in the aqueous phase of the vessels was measured using ionchromatography. Increasing time results in progressively higher levelsof nitrate in the sterile aquifer water thus demonstrating theeffectiveness of the invention for allowing gradual diffusion of aninorganic microbial nutrient through the porous surface.

EXAMPLE 2

A chart is shown in FIG. 4, which depicts the quantity of biomassobtained from individual porous surfaces of the appartus shown in FIG.2. after a 12 day in situ aquifer experiment. The designations fornutrients in the different component vessels of the apparatus of FIG. 1are as shown in FIG. 4 are as follows: C1 and C2: both control withoutnutrients; N1: 1500 mg nitrate/L; N10. 15000 mg nitrate/L; P1: 93 mgtrimetaphosphate/L; P10: 930 mg trimetaphosphate/L; Ac1: 100 mgacetate/L; Ac10: 1000 mg acetate/L; A3: acetate, nitrate andtrimetaphosphate added in 1000:143:9 C:N:P ratios; NA: nutrient agar.With the exception of nitrate, treatments with lower concentrations ofinorganic or organic nutrients allowed only limited quantities ofbiomass to develop on the porous surfaces. Control components exhibitedmarkedly lower levels of attached biomass than all other components,although P1 (93 mg trimetaphosphate/L) and Ac1 (100 mg acetate/L) werecomparable. It is not possible to distinguish between samples thatcontained the highest concentration of nutrients (N10, P10, AC10, A3 andNA) and there is an indication that in this aquifer microorganisms arelimited mainly by nitrate since even the lowest concentration of nitrate(N1 at 1500 mg nitrate/L) caused a significant increase in biomass abovecontrols. These results provide evidence that subsurface microorganismswhich become attached to the porous surfaces respond favorably tonutrient enrichment.

EXAMPLE 3

FIG. 5 shows a chart depicting the results of a multiple carbon sourceutilization assay conducted on microbial communities obtained from theporous surfaces of the apparatus depicted in FIG. 1 compared to theresults of the same assay performed on free-living microbial communitiesfrom the same aquifer. The data obtained for the utilization of 95different organic carbon sources by the microbial communities wasanalyzed using principal component analysis. These data indicate thatthe porous surface upon which cells adhere allows the development of amicrobial community unique from that in the liquid phase. The resultsalso suggest that the microbial communities that were attached to theporous surface of the present embodiment of the invention preferred touse amino acids and carboxylic acids relative to the free-livingmicroorganisms from the same aquifer which preferred to usecarbohydrates. Attached microbial communities in aquifers arenumerically and functionally dominant and the invention described hereinis designed to select for these communities as distinct from prior art.

We claim:
 1. A method of determining nutrient limitations on growth ofmicroorganisms in an aqueous environment comprising the steps of:a.providing a device comprisingi. holding means for holding a liquidnutrient medium for stimulating growth of said microorganisms, saidholding means including nutrient-diffusing substrate means disposed onsaid holding means for gradually releasing an effective amount of saidliquid nutrient medium such that said microorganisms are stimulated toattach to and grow on said nutrient-diffusing substrate means; and ii.suspension means disposed on said holding means for suspending saiddevice in said aqueous environment; b. placing a liquid nutrient mediumto be tested in said holding means; c. suspending said device in saidaqueous environment for a sufficient time for said nutrient-diffusingsubstrate means to gradually release said liquid nutrient medium suchthat said microorganisms attach to and grow on said nutrient-diffusingsubstrate means; and d. measuring growth of said microorganisms on saidnutrient-diffusing substrate means.
 2. The method of claim 1 whereinsaid device comprises a plurality of said holding means coupled togetherand spaced apart in a linear series by connecting means.
 3. The methodof claim 2 further comprising placing a different liquid nutrient mediumin each of said plurality of holding means and(e) comparing the growthmeasured in step (d) for each said different liquid nutrient medium. 4.The method of claim 3 wherein said device further comprises a gas sensordisposed on each said holding means for monitoring a gas given off bysaid microorganisms attached to and growing on each sidenutrient-diffusing substrate means.
 5. The method of claim 4 whereinsaid gas sensor monitors oxygen gas.
 6. The method of claim 4 whereinsaid device further comprises a computer linked to said gas sensor forreceiving monitoring data generated by said gas sensor and saidcomparing step comprises comparing said monitoring data.
 7. The methodof claim 5 wherein said gas sensor monitors carbon dioxide gas.
 8. Themethod of claim 5 wherein said gas sensor monitors a mixture of oxygengas and carbon dioxide gas.
 9. The method of claim 2 wherein saidholding means comprises an impermeable cylindrical side wall, animpermeable top disposed on said side wall, and a porous bottom disposedon said side wall, said porous bottom comprising said nutrient-diffusingsubstrate means and said side wall, top, and bottom defining a cavityfor holding said liquid nutrient medium.
 10. The method of claim 9wherein said device further comprises means for filling said cavity withsaid liquid nutrient medium.
 11. The method of claim 10 wherein saidfilling means comprises a removable plug.
 12. The method of claim 9wherein said porous bottom is detachable from said side wall and whereinsaid device further comprises means for sealing said porous bottom tosaid side wall.
 13. The method of claim 1 wherein saidnutrient-diffusing substrate means comprises a porous ceramic support.14. The method of claim 1 wherein said aqueous environment comprises anaquifer.
 15. A method of determining the nutrients to stimulatemicroorganisms in a particular environment comprising the steps of:a.contacting a representative sample of said microorganisms from saidenvironment with multiple support means wherein each said support meanshas intimately associated with the surface of said support means adifferent nutrient composition for said microorganisms in said sample,said support means comprising a container for said nutrient compositioncomprising a porous covering layer separating said nutrient compositionand said microorganisms which porous covering layer allows interactionwith said nutrient composition and said microorganisms at themicroorganism side of said porous layer; b. allowing said multiplesupport means to remain in contact with said microorganisms in saidsample for a time period sufficient to measure differences inmicroorganism effects for said multiple support means; and c. measuringand comparing microorganism effects for said multiple support means. 16.The method as in claim 15 wherein said nutrient composition passesthrough said porous covering layer by diffusion.
 17. The method of claim16 wherein said porous layer comprises a porous ceramic support.
 18. Themethod as in claim 15 wherein at least some of said support meanscomprise a cylindric shaped container having both ends of the cylindersealed so as to contain said nutrient composition and at least one ofsaid surfaces comprising a porous support.
 19. The method as in claim 18wherein each of said support means are connected to each other to form achain of support means.
 20. The method as in claim 15 wherein saidrepresentative sample is part of said particular environment itself andwherein said method is conducted in situ.
 21. The method as in claim 15wherein said measuring and comparing step (c) comprises subsequent tostep (b) measuring and comparing the quantity and type of microorganismgrowth on each said support.
 22. The method as in claim 15 comprising,responsive to the measuring and comparing step (c), the additional stepof regulating the quantity and type of said nutrient composition toproduce a desired effect upon said microorganisms in said particularenvironment.
 23. The method of claim 22 wherein the method is part of anin situ biological process wherein sad nutrient composition is adjustedto optimize the desired result.
 24. The method of claim 22 wherein themethod is part of a bioremediation process wherein said nutrientcomposition is adjusted to enhance the removal of the targeted wastecomponent.